Devices, methods, and kits for multiplexing a fluid sample via fluid sample reuse

ABSTRACT

Devices, methods, and kits for detecting at least two analytes present within a small volume single fluid sample obtained from patient for the creation of a multiplexed panel of the various analytes present within the single fluid sample are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/351,530, filed Jun. 17, 2016, the disclosure of which is herebyincorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

Not Applicable.

TECHNICAL FIELD

The presently disclosed and claimed inventive concept(s) relate to adevice(s) and method(s) for the detection of at least two analytes, saidanalytes being of different and distinct compositions, present within asmall volume of a single fluid sample. As a result of such detection, amultiplexed panel of the various analytes present within the smallvolume of the single fluid sample is achieved, while mitigating singlefluid sample loss/reduction and allowing for re-use of the single fluidsample.

BACKGROUND

Numerous devices and methods exist for detecting analytes that may bepresent in a fluid sample. However, the currently employed devices andmethods require the use of multiple fluid samples and/or aliquots of asingle fluid sample for the detection of analytes present in suchsample(s). Such devices and methods require a large volume of the fluidsample, the collection of multiple fluid samples from a patient in orderto achieve analyte detection, and/or result in fluid sampleloss/reduction due to multiple aliquots being taken from the fluidsample for analyte detection. Accordingly, a need exists for new andimproved devices and methods of high sensitivity that allow a smallvolume of a single fluid sample obtained from a patient to be re-used inorder to detect at least two analytes present in the single fluidsample. Such devices and methods thereby allow for the creation of amultiplexed panel associated with the various analytes present in thesingle fluid sample. It is to such devices and methods, as well as kitsrelated thereto, that the presently disclosed and claimed inventiveconcept(s) is directed.

DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are detailed perspective views of one embodiment ofa device constructed in accordance with the presently disclosed andclaimed inventive concept(s) and use thereof in a method as describedherein.

FIGS. 2A-2L are perspective views of an alternate embodiment of a deviceof the presently disclosed and claimed inventive concept(s) and usethereof in a method as described herein.

DETAILED DESCRIPTION

Before explaining at least one embodiment of the inventive concept(s) indetail by way of exemplary drawings, experimentation, results, andlaboratory procedures, it is to be understood that the inventiveconcept(s) is not limited in its application to the details ofconstruction and the arrangement of the components set forth in thefollowing description or illustrated in the drawings, experimentationand/or results. The inventive concept(s) is capable of other embodimentsor of being practiced or carried out in various ways. As such, thelanguage used herein is intended to be given the broadest possible scopeand meaning; and the embodiments are meant to be exemplary—notexhaustive. Also, it is to be understood that the phraseology andterminology employed herein is for the purpose of description and shouldnot be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed and claimed inventive concept(s)shall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Enzymatic reactions and purification techniquesare performed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. The nomenclatures utilized in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well-known and commonly used in the art.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this presently disclosed and claimedinventive concept(s) pertains. All patents, published patentapplications, and non-patent publications referenced in any portion ofthis application are herein expressly incorporated by reference in theirentirety to the same extent as if each individual patent or publicationwas specifically and individually indicated to be incorporated byreference.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this presentlydisclosed and claimed inventive concept(s) have been described in termsof preferred embodiments, it will be apparent to those of skill in theart that variations may be applied to the compositions and/or methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of thepresently disclosed and claimed inventive concept(s). All such similarsubstitutes and modifications apparent to those skilled in the art aredeemed to be within the spirit, scope and concept of the inventiveconcept(s) as defined by the appended claims.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The singular forms “a,” “an,” and “the”include plural referents unless the context clearly indicates otherwise.Thus, for example, reference to “a compound” may refer to 1 or more, 2or more, 3 or more, 4 or more or greater numbers of compounds. The term“plurality” refers to “two or more.” The use of the term “or” in theclaims is used to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects. For example but not byway of limitation, when the term “about” is utilized, the designatedvalue may vary by ±20% or ±10%, or ±5%, or ±1%, or ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods and as understood by persons having ordinary skill inthe art. The use of the term “at least one” will be understood toinclude one as well as any quantity more than one, including but notlimited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “atleast one” may extend up to 100 or 1000 or more, depending on the termto which it is attached; in addition, the quantities of 100/1000 are notto be considered limiting, as higher limits may also producesatisfactory results. In addition, the use of the term “at least one ofX, Y and Z” will be understood to include X alone, Y alone, and Z alone,as well as any combination of X, Y and Z. The use of ordinal numberterminology (i.e., “first”, “second”, “third”, “fourth”, etc.) is solelyfor the purpose of differentiating between two or more items and is notmeant to imply any sequence or order or importance to one item overanother or any order of addition, for example.

As used in this specification and claim(s), the terms “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “substantially” means that the subsequentlydescribed event or circumstance completely occurs or that thesubsequently described event or circumstance occurs to a great extent ordegree. For example, the term “substantially” means that thesubsequently described event or circumstance occurs at least 90% of thetime, or at least 95% of the time, or at least 98% of the time.

As used herein, the phrase “associated with” includes both directassociation of two moieties to one another as well as indirectassociation of two moieties to one another. Non-limiting examples ofassociations include covalent binding of one moiety to another moietyeither by a direct bond or through a spacer group, non-covalent bindingof one moiety to another moiety either directly or by means of specificbinding pair members bound to the moieties, incorporation of one moietyinto another moiety such as by dissolving one moiety in another moietyor by synthesis, and coating one moiety on another moiety.

The term “purified” as used herein means at least one order of magnitudeof purification is achieved compared to the starting material or ofmaterial in its natural state, for example but not by way of limitation,two, three, four or five orders of magnitude of purification of thestarting material or of the natural material. Thus, the term “purified”as utilized herein does not necessarily mean that the material is 100%purified, and therefore such term does not exclude the presence of othermaterial(s) present in the purified composition.

The term “sample” as used herein will be understood to include any typeof biological fluid sample that may be utilized in accordance with thepresently disclosed and claimed inventive concept(s). Examples ofbiological samples that may be utilized include, but are not limited to,whole blood or any portion thereof (i.e., plasma or serum), saliva,sputum, cerebrospinal fluid (CSF), intestinal fluid, intraperotinealfluid, cystic fluid, sweat, interstitial fluid, tears, mucus, urine,bladder wash, semen, combinations, and the like. The volume of thesample utilized in accordance with the presently disclosed and claimedinventive concept(s) is from about 1 to about 100 microliters. As usedherein, the term “small volume” as it relates to the sample utilized inaccordance with the presently disclosed and claimed inventive concept(s)means from about 0.1 microliter to about 90 microliters, or from about 1microliter to about 75 microliters, or from about 2 microliters to about60 microliters, or less than or equal to about 50 microliters.

The term “patient” includes human and veterinary subjects. In certainembodiments, a patient is a mammal. In certain other embodiments, thepatient is a human. “Mammal” for purposes of treatment refers to anyanimal classified as a mammal, including human, domestic and farmanimals, nonhuman primates, and zoo, sports, or pet animals, such asdogs, horses, cats, cows, etc.

Turning now to particular embodiments of the presently claimed anddisclosed inventive concept(s), devices, methods, and kits are hereindisclosed of high sensitivity that allow for a small volume of a singlefluid sample obtained from a patient to be re-used in order to detect atleast two analytes present in the single fluid sample.

The kit can further include a set of written or pictorial instructions(or information on how to obtain instructions, either written orpictorial, from the internet) explaining how to use the kit. A kit ofthis nature can be used in any of the methods described or otherwisecontemplated herein.

It is contemplated that virtually any reagent used in the fields ofbiological, chemical, or biochemical analyses could be used in thedevices, kits, and methods of the presently claimed and disclosedinventive concept(s). It is contemplated that these reagents may undergophysical and/or chemical changes when bound to an analyte of interestwhereby the intensity, nature, frequency, or type of signal generated bythe reagent-analyte complex is proportional to the concentration of theanalyte existing within the fluid sample. These reagents may containindicator dyes, metal, enzymes, polymers, antibodies, andelectrochemically reactive ingredients.

Any method of detecting and measuring the analyte in a fluid sample canbe used in the devices, kits, and methods of the presently claimed andinventive concepts. A variety of assays for detecting analytes are wellknown in the art and include, but are not limited to, enzyme inhibitionassays, antibody stains, latex agglutination, and immunoassays, such as,radioimmunoassays. The term “antibody” herein is used in the broadestsense and refers to, for example, intact monoclonal antibodies,polyclonal antibodies, multi-specific antibodies (e.g., bispecificantibodies), and to antibody fragments that exhibit the desiredbiological activity (e.g., antigen/analyte-binding). The antibody can beof any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

The term “binding partner” as used herein will be understood to refer toany molecule capable of associating with another molecule. For example,but not by way of limitation, the binding partner may be an antibody(including polyclonal or monoclonal antibodies), antibody fragments(such as, but not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd,diabodies, single-chain antibodies, and other antibody fragments thatretain at least a portion of the variable region of the intactantibody), a receptor, a ligand, aptamers, antibody substitute proteinsor peptides (i.e., engineered binding proteins/peptides), molecularimprinted polymers (i.e., inorganic matrices), combinations orderivatives thereof, as well as any other molecules capable of bindingto the analyte(s).

The term “non-reacted portion” as used herein will be understood torefer to any portion of the single fluid sample in which an analyte hasnot become associated with an immobilized capture antibody/bindingpartner present on an inner surface of a pipette and which isre-dispensed into the sample receptacle. The term “reacted portion” asused herein will be understood to refer to any portion of the singlefluid sample in which an analyte has become associated with animmobilized capture antibody/binding partner on an inner surface of apipette and which is not re-dispensed into the sample receptacle.

While immunoassays are primarily discussed herein, a person havingordinary skill in the art should readily understand that the presentlydisclosed and claimed inventive concept(s) are not strictly limited toimmunoassays and may include, by way of example and not by limitation,nucleic acid capture assays and serology-based assays. Immunoassays,including radioimmunoassays and enzyme-linked immunoassays, are usefulmethods for use with the presently claimed and disclosed inventiveconcepts. A variety of immunoassay formats, including, for example,competitive and non-competitive immunoassay formats, antigen/analytecapture assays and two-antibody sandwich assays can be used in themethods of the invention. Enzyme-linked immunosorbent assays (ELISAs)can be used in the presently claimed and disclosed inventive concepts,as well. In the case of an enzyme immunoassay, an enzyme is typicallyconjugated to a second antibody, generally by means of glutaraldehyde,periodate, hetero-bifunctional crosslinking agents, orbiotin-streptavidin complexes. As will be readily recognized, however, awide variety of different conjugation techniques exist which are readilyavailable for use with the presently disclosed and claimed inventiveconcept(s) to one skilled in the art.

In certain embodiments, the analytes are detected and measured usingchemiluminescent detection. For example, in certain embodiments,analyte-specific capture antibodies are used to capture an analytepresent in the biological fluid sample. The analyte-specific captureantibodies (and/or the specific analyte(s)) may be labeled with achemiluminescent detection antibody to thereby detect if an analyte ispresent in the biological fluid sample. Any chemiluminescent label anddetection system can be used in the present devices and methods.Chemiluminescent detection antibodies/binding partners can be obtainedcommercially from various sources. Methods of detecting chemiluminescentdetection antibodies/binding partners are known in the art and are notfurther discussed here in detail.

Fluorescent detection also can be useful for detecting analytes in thepresently claimed and disclosed inventive concepts. Useful fluorochromesinclude, but are not limited to, DAPI, fluorescein, lanthanide metals,Hoechst 33258, R-phycocuanin, B-phycoerythrin, R-phycoerythrin,rhodamine, Texas red and lissamine. Fluorescent compounds can bechemically coupled to antibodies/binding partners without altering theirbinding capacity. When activated by illumination with light of aparticular wavelength, the fluorochrome-labeled antibody/binding partnerabsorbs the light energy, inducing a state of excitability in themolecule, followed by emission of the light as a characteristic colorthat is visually detectable. Radioimmunoassays (RIAs) can also be usefulin certain methods of the invention. Such assays are well known in theart. Radioimmunoassays can be performed, for example, with ¹²⁵I-labeleddetection antibodies/binding partners that are associated with eitherthe analyte being detected and/or the immobilized captureantibodies/binding partners.

Assays, including, but not limited to, immunoassays, nucleic acidcapture assays, and serology-based assays, can be developed for amultiplexed panel of proteins, peptides, and nucleic acids which may becontained within a fluid sample, with such proteins and peptidesincluding, for example but not by way of limitation, albumin,hemoglobin, myoglobin, α-1-microglobin, immunoglobins, enzymes,glycoproteins, protease inhibitors, drugs, and cytokines. The device maybe used for the analysis of any fluid sample, including, withoutlimitation, whole blood, plasma, serum, or urine. Table 1 below, by wayof example only and not by way of limitation, illustrates particularanalytes of interest as well as respective antibodies/binding partnerswhich may be used in the presently disclosed and claimed inventiveconcept(s).

TABLE 1 Non-Limiting Examples of Analytes and Antibodies/BindingPartners 25-hydroxyvitamin D; 1,25 dihydroxyvitamin D - serum/plasmaAcetaminophen - serum/plasma ACTH - serum/plasma AFP - serum ALB -serum/plasma Alpha-1-Antitrypsin blood SERPINA 1 Alpha-1-Antitrypsinserum/plasma Alpha-1-Acid Glycoprotein serum/plasma Amphetamines - urineAmylase - pancreatitis blood, urine or peritioneal sample ANA -autoimmune (SLE) serum/plasma Androstenedione serum/plasmaAnti-double-stranded DNA, IgG -serum Apolipoprotein A-I - serum/plasmaBarbituates - urine Benzodazepines - serum/plasma Benzodazepines - urineBeta2 Microglobulin - serum or 24 h urine Blood Typing (Immunoassay) -Panel/Whole Blood BNP - plasma Borrelia burgdorferi Antibodies (LymeDisease) - serum/plasma CA -125 - serum/plasma CA 15-3 - serum/plasma CA19-9 - serum/plasma CA 27.29 - serum/plasma Calcitonin - serum/plasmaCarbamazepine - serum/plasma Cardiac-specific Troponin I and TroponinT - serum/plasma Cardiolipin Antibodies (IgG) - serum/plasma CEA -serum/plasma Celiac Panel (4 plex) - serum/plasma Chlamydia trachomatisDNA Qualitative Chlamydia trachomatis DNA Qualitative - Swab CollectionChlamydia Trachomatis, DNA, Qualitative/Gonorrhea, DNA, QualitativeCholinesterase - serum/plasma CKMB - serum/plasma CMV Antibody IgG -serum/plasma CMV Antibody IgM - serum/plasma Cocaine - urine CollagenCrosslinks - serum/plasma Cortisol, Total - serum/plasma C-peptide(insulin) - serum/plasma CRP -serum/plasma Cyclic Citrullinated Peptide(CCP) Antibody, IgG - serum/plasma Cyclosporine, A (immunosupressant) -serum/plasma Cystatin C (kidney) - serum/plasma D-Dimer -serum/plasmaDeaminated Gliadin Peptide IgA -serum/plasma Deaminated Gliadin PeptideIgG -serum/plasma Dehydroepiandrosterone Sulfate - serum/plasmaDehydroepiandrosterone Sulfate - serum/plasma GC/MS Digoxin -serum/plasma TDM Dolophine (Methadone) Double Stranded DNA (Crithidialuciliae) Antibody, IgG E. coli Shiga Toxin EBV Antibody (NA IgG) -serum EBV Antibody Panel (NA IgG; VCA IgG; VCA IgM) - serum EBV AntibodyPanel (VCA IgG) - serum EBV Antibody Panel (VCA IgM) - serum Ecstasy(MDMA) Endomysial Antibody; EMA IgA - serum (ELISA and IFA) EndomysialAntibody; EMA IgG - serum (ELISA and IFA) Estradiol - serum Estriol -serum/plasma Estriol - urine (24 hour collection) Ferritin - serumFibrinogen - plasma Folate - serum Follicle Stimulating Hormone -serum/plasma Follicle Stimulating Hormone - urine Gastrin - serumGentamicin - serum Growth Hormone - serum H. pylori, IgG - serumHaptoglobin, Quantitative - serum hCG - serum/plasma hCG - urineHepatitis A Antibody (HAAb), Total - serum Hepatitis A, IgM - serumHepatitis B Core Antibody, IgM - serum Hepatitis B Core Antibody,Total - serum Hepatitis B Surface Antibody, Total - serum Hepatitis B,DNA, Quantitative Hepatitis C Antibody - serum Hepatitis C GenotypingHER2/neu, Quantitative - serum Herpes Simplex Virus, Type 1, IgG - serumHIV-1/2 Ab - serum Homocysteine - serum/plasma hsCRP - serum/plasmaHuman Immunodeficiency Virus 1, RNA Viral Load, Quantitative HumanImmunodeficiency Virus Antibody Test - serum IgA, serum IgE IgG, serumIgM, serum Insulin - serum/plasma Lactate Dehydrogenase, Total andIsoenzymes - Body Fluid/Serum Lipase - serum Luteinizing Hormone - serumLuteinizing Hormone - urine Measles (Rubeola Antibody), IgG - serum/CSFMethadone Metabolite - urine Microalbumin - urine Mumps Antibody, IgG -serum Myoglobin - serum/plasma Neisseria gonorrhoeae, DNA, QualitativeNuclear Antigen Antibody, Jo-1 - serum Nuclear Antigen Antibody, RNP -serum Nuclear Antigen Antibody, Scl-70 - serum Nuclear Antigen Antibody,Sm - serum Nuclear Antigen Antibody, SSA - serum Nuclear AntigenAntibody, SSB - serum Opiates - urine Parathyroid Hormone - serumPhencyclidine (PCP) - urine Phenobarbital - serum/plasma Phenytoin,Total - serum/plasma Progesterone - serum Prolactin - serum PropoxyphenePSA, Free - serum/plasma PSA, Total - serum RBC Antibody ID(Immunoassay) - Whole Blood Rheumatoid Factor, Total - serum RubellaAntibodies, IgG - serum Rubella Antibodies, IgM - serum Salicylate SexHormone-binding Globulin (SHBG) Sex Hormone-binding Globulin (SHBG) -serum/plasma Streptolysin O Antibody, Titer (ASO) - serum SyphilisScreen (Treponema Pallidum Antibody) - serum T3, Free -Triiodothyronine, Free - serum T3, Total - Triiodothyronine, Total -serum T4, Free - Thyroxine, Free - serum T4, Total - Thyroxine, Total -serum Testosterone, Free - serum/plasma Testosterone, Total -serum/plasma THC - urine Theophylline - serum Thyroglobulin - serum orplasma Thyroglobulin Antibodies - serum Thyroid Peroxidase Antibody(TPO) Thyroid Stimulating Hormone (TSH) - serum Thyroxine BindingGlobulin - serum Tobramycin -serum/plasma Toxoplasma, IgG - serumToxoplasma, IgM - serum Tricyclic Antidepressants - urine TricyclicAntidepressants, Blood Triiodothyronine Uptake (T3 Uptake) - serumValproic Acid - serum/plasma Vancomycin - serum Varicella-ZosterAntibody - serum Viral Hepatitis C, RNA, Quantitative Vitamin B12 -serum

In addition and as illustrated in Table 1 above, while the Figuresspecifically show immobilized capture antibodies present on the innersurface(s) of the pipette(s), it should be readily understood by aperson having ordinary skill in the art that any binding partner may beimmobilized on the inner surface(s) of the pipette(s) to accomplish theinventive concept(s), including, by way of example and not bylimitation, nucleic acids and fragments thereof, antigens, ligands, andany binding partner capable of accomplishing the presently disclosed andclaimed inventive concept(s).

Referring now to the drawings, and more particularly to FIGS. 1A-1C,shown therein is an exemplary embodiment of a device 10 that detects thepresence and/or absence of at least two analytes in a single fluidsample obtained from a patient. The device 10 comprises a samplereceptacle 12 and at least one pipette 14, i.e., the device 10 maycontain 1, 2, 5, 10, 15, 20, 50, 100, 100 or 1000 pipettes attached toone another via a mechanical connecting web or a pipetting station, suchas pipetting station 15 for example, but not by way of limitation. Byway of example only, FIGS. 1A-1C depict two pipettes shown as 14 and 26to illustrate that the device 10 may comprise more than one pipette. Forpurposes of clarity only and not by way of limitation, the device 10shown in FIGS. 1A-1C shall be described with reference only to pipette14. Pipette 14 comprises an inner surface 16 on which at least oneimmobilized capture antibody 20 is present. The sample receptacle 12contains a single fluid sample 18 that has been obtained from a patient.As shown in FIG. 1A with respect to pipette 14, five immobilized captureantibodies 20 are shown as being immobilized on the inner surface 16.However, one of ordinary skill in the art would recognize that anynumber of immobilized capture antibodies 20 may be immobilized on theinner surface 16 of the pipette 14.

As depicted in FIG. 1B, at least a portion of the single fluid sample 18has been withdrawn into the pipette 14 such that the single fluid sample18 is in contact with the inner surface 16 of the pipette 14. As aresult of this contact, the single fluid sample 18 comes into contactwith immobilized capture antibody/binding partner 20. If an analyte ofinterest is present in the single fluid sample 18, at least oneanalyte-antibody/binding partner complex 24 is formed by an associationbetween the analyte 22 present in the single fluid sample 18 and the atleast one immobilized capture antibody/binding partner 20 present on theinner surface 16 of the pipette 14.

As shown in FIG. 1C, a non-reacted portion of the single fluid sample 18is re-dispensed into the sample receptacle 12 from the pipette 14. Theanalyte 22, if present, remains within the pipette 14 (a reacted portionof the single fluid sample 18) due to the association between theanalyte 22 and the immobilized capture antibody 20 forming the firstanalyte-antibody/binding partner complex 24 present on the inner surface16 of the pipette 14. Detection of the analyte 22 within the pipette 14can be accomplished in accordance with any of the methodologiesdescribed herein or well known in the art. The detection can be either aqualitative or quantitative assessment of the presence of analyte 22within the pipette 14 and thereby indicates the presence of analyte 22in the single fluid sample 18.

Thereafter, pipette 14 is removed from the sample receptacle 12 andpipette 26 is placed within the sample receptacle 12. Thereafter, atleast a portion of the non-reacted portion of the single fluid sample 18is withdrawn into pipette 26 and into contact with an inner surface 28of pipette 26 to which a second immobilized capture antibody 30 isbound. A second analyte (not shown) which is different in compositionthan the analyte 22 is thereafter, if present in the single fluid sample18, bound to the second immobilized capture antibody/binding partner 30bound to inner surface 28 of pipette thereby forming a secondanalyte-antibody/binding partner complex (not shown).

Referring now to FIGS. 2A-2L, shown therein is an alternate embodimentof a device 10A that detects the presence and/or absence of at least twoanalytes in a single fluid sample obtained from a patient. The device10A comprises a sample receptacle 12A, a first pipette 14A, a secondpipette 26A, and a third pipette 36A. While the non-limiting embodimentof device 10A as shown in FIGS. 2A-2L is shown as comprising threepipettes, three analytes, and three distinct and different immobilizedcapture antibodies/binding partners, it should be understood to a personhaving ordinary skill in the art that device 10A can be comprised of anynumber of pipettes, analytes, and different and distinct immobilizedcapture antibodies/binding partners in order to detect the presence ofat least two analytes present within a single fluid sample obtained froma patient. For example, the device 10A may contain 1, 2, 5, 10, 15, 20,50, 100, or 1000 pipettes. The pipettes may be attached to one anothervia a mechanical connecting web or pipetting station, such as pipettingstation 15A for example, but not by way of limitation. In addition, theprocess of sequentially disposing the first pipette 14A, the secondpipette 26A, and the third pipette 36A within the sample receptacle 12Acan be accomplished either by a manual or automated process(es) via useof the pipetting station 15A.

As shown in FIGS. 2A-2D, the first pipette 14A comprises an innersurface 16A on which a first immobilized capture antibody 20A ispresent. As shown in FIGS. 2A-2D, five first immobilized captureantibodies/binding partners 20A are shown as being immobilized on theinner surface 16A. However, one of ordinary skill in the art wouldrecognize that any number of first immobilized captureantibodies/binding partners 20A may be immobilized on the inner surface16A of the first pipette 14A. The first pipette 14A is disposed withinsample receptacle 12A which contains a single fluid sample 18A obtainedfrom a patient, said single fluid sample 18A comprising at least twoanalytes. At least a portion of the single fluid sample 18A is withdrawninto the first pipette 14A such that the single fluid sample 18A is incontact with the inner surface 16A of the first pipette 14A. As a resultof this contact, the single fluid sample 18A comes into contact with thefirst immobilized capture antibody/binding partner 20A. If a firstanalyte of interest is present in the single fluid sample 18A, at leastone first analyte-antibody/binding partner complex 24A is formed by anassociation between the first analyte 22A present in the single fluidsample 18A and the first immobilized capture antibody 20A present on theinner surface 16A of the first pipette 14A. A non-reacted portion of thesingle fluid sample 18A is re-dispensed into the sample receptacle 12Afrom the first pipette 14A. The first analyte 22A, if present, remainswithin the first pipette 14A (a reacted portion of the single fluidsample 18A) due to the association between the first analyte 22A and thefirst immobilized capture antibody/binding partner 20A forming the firstanalyte-antibody complex 24A present on the inner surface 16A of thefirst pipette 14A. Detection of the first analyte 22A within the firstpipette 14A can be accomplished in accordance with any of themethodologies described herein or well known in the art. The detectioncan be either a qualitative or quantitative assessment of the presenceof the first analyte 22A within the pipette 14A and thereby indicativeof the presence of the first analyte 22A in the single fluid sample 18A.After the non-reacted portion of the single fluid sample 18A isre-dispensed into the sample receptacle 12A from the first pipette 14A,the second pipette 26A is transitioned so that is capable of beingdisposed within the sample receptacle 12A.

Referring now to FIGS. 2E-2H, the second pipette 26A comprises an innersurface 28A on which a second immobilized capture antibody/bindingpartner 30A is present. The second immobilized capture antibody/bindingpartner 30A is different in analyte specificity from the firstimmobilized capture antibody/binding partner 20A, thereby allowing forassociation with and detection of a different and distinct analytepresent in the single fluid sample 18A from the first analyte 22Apresent in the single fluid sample 18A. The second pipette 26A isdisposable within sample receptacle 12A which contains the non-reactedportion of the single fluid sample 18A which has been re-dispensed intothe sample receptacle 12A from the first pipette 14A. As shown in FIGS.2E-2H, with respect to the second pipette 26A, five second immobilizedcapture antibodies/binding partners 30A are shown as being immobilizedin the inner surface 28A. However, one of ordinary skill in the artwould recognize that any number of second immobilized captureantibodies/binding partners 30A may be immobilized on the inner surface28A of the second pipette 26A. At least a portion of the non-reactedportion of the single fluid sample 18A is withdrawn into the secondpipette 26A such that the single fluid sample 18A is in contact with theinner surface 28A of the second pipette 26A. As a result of thiscontact, the single fluid sample 18A comes into contact with the secondimmobilized capture antibody/binding partner 30A. If a second analyte ofinterest is present in the single fluid sample 18A, at least one secondanalyte-antibody/binding partner complex 34A is formed by an associationbetween the second analyte 32A present in the single fluid sample 18Aand the second immobilized capture antibody/binding 30A present on theinner surface 28A of the second pipette 26A. A non-reacted portion ofthe single fluid sample 18A is re-dispensed into the sample receptacle12A from the second pipette 26A. The second analyte 32A, if present,remains within the second pipette 26A (a reacted portion of the singlefluid sample 18A) due to the association between the second analyte 32Aand the second immobilized capture antibody/binding partner 30A formingthe second analyte-antibody/binding partner complex 34A present on theinner surface 28A of the second pipette 26A. Detection of the secondanalyte 32A within the second pipette 26A can be accomplished inaccordance with any of the methodologies described herein or well knownin the art. The detection can be either a qualitative or quantitativeassessment of the presence of the second analyte 32A within the secondpipette 26A and thereby indicative of the presence of the second analyte32A in the single fluid sample 18A=. After the non-reacted portion ofthe single fluid sample 18A is re-dispensed into the sample receptacle12A, the third pipette 36A is transitioned so that it is capable ofbeing disposed within the sample receptacle 12A.

Referring now to FIGS. 21-2L, the third pipette 36A comprises an innersurface 38A on which a third immobilized capture antibody/bindingpartner 40A is present. The third immobilized capture antibody/bindingpartner 40A is different in analyte specificity from the firstimmobilized capture antibody/binding partner 20A and the secondimmobilized capture antibody/binding partner 30A, thereby allowing forassociation with and detection of a different and distinct analytepresent in the single fluid sample 18A from the first analyte 22A andsecond analyte 32A present in the single fluid sample 18A. The thirdpipette 36A is disposed within sample receptacle 12A which contains thenon-reacted portion of the single fluid sample 18A which has beenre-dispensed into the sample receptacle 12A from the second pipette 26A.As shown in FIGS. 21-2L, with respect to the third pipette 36A, fivethird immobilized capture antibodies/binding partners 40A are shown asbeing immobilized on the inner surface 38A. However, one of ordinaryskill in the art would recognize that any number of third immobilizedcapture antibodies/binding partners 40A may be immobilized on the innersurface 38A of the third pipette 36A. At least a portion of thenon-reacted portion of the single fluid sample 18A is withdrawn into thethird pipette 36A such that the single fluid sample 18A is in contactwith the inner surface 38A of the third pipette 36A. As a result of thiscontact, the single fluid sample 18A comes into contact with the thirdimmobilized capture antibody/binding partner 40A. If a third analyte ofinterest is present in the single fluid sample 18A, at least one thirdanalyte-antibody complex 44A is formed by an association between thethird analyte 42A present in the single fluid sample 18A and the thirdimmobilized capture antibody/binding partner 40A present on the innersurface 38A of the third pipette 36A. A non-reacted portion of thesingle fluid sample 18A is re-dispensed into the sample receptacle 12Afrom the third pipette 36A. The third analyte 42A, if present, remainswithin the third pipette 36A (a reacted portion of the single fluidsample 18A) due to the association between the third analyte 42A and thethird immobilized capture antibody/binding partner 40A forming the thirdanalyte-antibody complex 44A present on the inner surface 38A of thethird pipette 36A. Detection of the third analyte 42A within the thirdpipette 36A can be accomplished in accordance with any of themethodologies described herein or well known in the art. The detectioncan be either a qualitative or quantitative assessment of the presenceof the third analyte 42A within the third pipette 36A and therebyindicative of the presence of the third analyte 42A in the single fluidsample 18A.

Non-Limiting Examples of the Inventive Concept(s)

A device for detecting the presence of at least two analytes in a fluidsample, the device comprising a pipetting station, wherein the pipettingstation is configured to receive at least two pipettes, wherein the atleast two pipettes comprises a first pipette having a first immobilizedbinding partner specific to a first analyte present on an inner surfaceof the first pipette and a second pipette having a second immobilizedbinding partner specific to a second analyte present on an inner surfaceof the second pipette, wherein the analyte specificity of the firstimmobilized binding partner is different than the analyte specificity ofthe second immobilized binding partner; wherein the first pipette isdisposable within a fluid sample receptacle configured to hold a fluidsample, the first pipette configured to withdraw at least a portion ofthe single fluid sample into the first pipette such that the firstimmobilized binding partner is brought into contact with the withdrawnfluid sample to thereby form a first analyte-binding partner complex ifa first analyte is present in the fluid sample; and further wherein anon-reacted portion of the single fluid sample is re-dispensable fromthe first pipette into the fluid sample receptacle, and thereafter thesecond pipette is disposable within the fluid sample receptacle wherebythe second pipette is configured to withdraw at least a portion of thenon-reacted portion of the fluid sample such that the second immobilizedbinding partner is brought into contact with the non-reacted portion ofthe withdrawn fluid sample to thereby form a second analyte-bindingpartner complex if a second analyte is present in the non-reactedportion of the single fluid sample.

The fluid sample of the device is selected from the group consisting ofwhole blood, blood plasma, blood serum, saliva, sputum, cerebrospinalfluid (CSF), intestinal fluid, intraperotineal fluid, cystic fluid,sweat, interstitial fluid, tears, mucus, urine, bladder wash, and semen.

The fluid sample of the device comprises a volume from about 1microliter to about 100 microliters. The volume of the fluid sample ofthe device is about 50 microliters.

The sample receptacle of the device is selected from the groupconsisting of a well, cuvette, tube, vial, and capillary.

The first analyte and second analyte of the device are selected from thegroup consisting of cardiac markers, infectious disease serologicalanalytes, therapeutic drugs, drugs of abuse, hormones, cancer markers,infectious disease nucleic acid analytes, autoimmune disease nucleicacid markers, proteins, vitamins, cofactors, metabolites, andcombinations thereof.

The first immobilized binding partner and the second immobilized bindingpartner of the device are selected from the group consisting of intactmonoclonal antibodies, polyclonal antibodies, multi-specific antibodies,bispecific antibodies, antibody fragments, receptors, ligands, aptamers,antigens, antibody substitute proteins or peptides, molecular imprintedpolymers, and combinations thereof.

The first immobilized capture antibody and the second immobilizedcapture antibody of the device are selected from the group consisting ofIgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, andcombinations thereof.

A method for detecting the presence of at least two analytes in a fluidsample, the method comprising the steps of upon receipt of a fluidsample which may comprise an analyte, placing the single fluid sample ina sample receptacle; drawing the single fluid sample into a firstpipette that is disposed within the sample receptacle, wherein a firstimmobilized binding partner is present on the an inner surface of thefirst pipette and the first immobilized binding partner is brought intocontact with the fluid sample such that a first analyte-binding partnercomplex is formed if a first analyte is present in the single fluidsample; re-dispensing a non-reacted portion of the fluid sample from thefirst pipette and into the fluid sample receptacle; drawing thenon-reacted portion of the fluid sample into a second pipette that isdisposed within the sample receptacle, wherein a second immobilizedbinding partner having an analyte specificity different than the firstimmobilized binding partner is present on an inner surface of the secondpipette and the second immobilized binding partner is brought intocontact with the non-reacted portion of the fluid sample such that asecond analyte-binding partner complex is formed if a second analyte ispresent in the non-reacted portion of the fluid sample; and detectingthe presence of the first analyte-binding partner complex and thepresence of the second analyte-binding partner complex.

The fluid sample of the method is selected from the group consisting ofwhole blood, blood plasma, blood serum, saliva, sputum, cerebrospinalfluid (CSF), intestinal fluid, intraperotineal fluid, cystic fluid,sweat, interstitial fluid, tears, mucus, urine, bladder wash, and semen.

The fluid sample of the method comprises a volume from about 1microliter to about 100 microliters. The volume of the fluid sample ofthe device is about 50 microliters.

The sample receptacle of the method is selected from the groupconsisting of a well, cuvette, tube, vial, and capillary.

The first analyte and second analyte of the method are selected from thegroup consisting of cardiac markers, infectious disease serologicalanalytes, therapeutic drugs, drugs of abuse, hormones, cancer markers,infectious disease nucleic acid analytes, autoimmune disease nucleicacid markers, proteins, vitamins, cofactors, metabolites, andcombinations thereof.

The first immobilized binding partner and the second immobilized bindingpartner of the method are selected from the group consisting of intactmonoclonal antibodies, polyclonal antibodies, multi-specific antibodies,bispecific antibodies, antibody fragments, receptors, ligands, aptamers,antigens, antibody substitute proteins and peptides, molecular imprintedpolymers, and combinations thereof.

The first immobilized binding partner and the second immobilized bindingpartner of the method are selected from the group consisting of IgG,IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and combinationsthereof.

The detection of the first analyte and the second analyte of the methodis conducted via chemiluminescent detection, fluorescent detection, andcombinations thereof.

A kit for detecting the presence of at least two analytes in a fluidsample, the kit comprising at least two pipettes, wherein the at leasttwo pipettes comprises a first pipette having a first immobilizedcapture antibody specific to a first analyte present on an inner surfaceof the first pipette and a second pipette having a second immobilizedcapture antibody specific to a second analyte present on an innersurface of the second pipette, wherein the analyte specificity of thefirst immobilized capture antibody is different than the analytespecificity of the second immobilized capture antibody; a fluid samplereceptacle, wherein the fluid sample receptacle is configured to hold afluid sample; and providing instructions to a user to allow the user touse the kit to identify the presence of at least two analytes in thefluid sample.

Thus, in accordance with the presently disclosed and claimed inventiveconcept(s), there have been provided devices, methods, and kits thatallow a small volume of a single fluid sample obtained from a patient tobe re-used in order to detect at least two analytes present in suchsingle fluid sample. Accordingly, a multiplexed panel associated withthe various analytes present in the single fluid sample is created thatfully satisfy the objectives and advantages set forth hereinabove.Although the presently disclosed and claimed inventive concept(s) hasbeen described in conjunction with the specific drawings,experimentation, results and language set forth hereinabove, it isevident that many alternatives, modifications, and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications and variations that fallwithin the spirit and broad scope of the presently disclosed and claimedinventive concept(s).

1. A device for detecting the presence of at least two analytes in apatient's single fluid sample, the device comprising: a pipettingstation, wherein the pipetting station is configured to receive at leasttwo pipettes, wherein the at least two pipettes comprises a firstpipette having a first immobilized binding partner specific to a firstanalyte present on an inner surface of the first pipette and a secondpipette having a second immobilized binding partner specific to a secondanalyte present on an inner surface of the second pipette, wherein theanalyte specificity of the first immobilized binding partner isdifferent than the analyte specificity of the second immobilized bindingpartner; wherein the first pipette is disposable within a fluid samplereceptacle configured to hold a patient's single fluid sample, the firstpipette configured to withdraw at least a portion of the single fluidsample into the first pipette such that the first immobilized bindingpartner is brought into contact with the withdrawn single fluid sampleto thereby form a first analyte-binding partner complex if a firstanalyte is present in the single fluid sample; and further wherein anon-reacted portion of the single fluid sample is re-dispensable fromthe first pipette into the fluid sample receptacle, and thereafter thesecond pipette is disposable within the fluid sample receptacle wherebythe second pipette is configured to withdraw at least a portion of there-dispensed fluid sample such that the second immobilized bindingpartner is brought into contact with the re-dispensed fluid sample tothereby form a second analyte-binding partner complex if a secondanalyte is present in the re-dispensed fluid sample.
 2. The device ofclaim 1, wherein the single fluid sample is selected from the groupconsisting of whole blood, blood plasma, blood serum, saliva, sputum,cerebrospinal fluid (CSF), intestinal fluid, intraperotineal fluid,cystic fluid, sweat, interstitial fluid, tears, mucus, urine, bladderwash, and semen.
 3. The device of claim 1, wherein the patient's singlefluid sample comprises a volume from about 1 microliter to about 100microliters.
 4. The device of claim 3, wherein the volume of thepatient's single fluid sample is about 50 microliters.
 5. The device ofclaim 1, wherein the sample receptacle is selected from the groupconsisting of a well, cuvette, tube, vial, and capillary.
 6. The deviceof claim 1, wherein the first analyte and second analyte are selectedfrom the group consisting of cardiac markers, infectious diseaseserological analytes, therapeutic drugs, drugs of abuse, hormones,cancer markers, infectious disease nucleic acid analytes, autoimmunedisease nucleic acid markers, proteins, vitamins, cofactors,metabolites, and combinations thereof.
 7. The device of claim 1, whereinthe first immobilized binding partner and the second immobilized bindingpartner are selected from the group consisting of intact monoclonalantibodies, polyclonal antibodies, multi-specific antibodies, bispecificantibodies, antibody fragments, receptors, ligands, aptamers, antigens,antibody substitute proteins or peptides, molecular imprinted polymers,and combinations thereof.
 8. The device of claim 7, wherein the firstimmobilized capture antibody and the second immobilized capture antibodyare selected from the group consisting of IgG, IgE, IgM, IgD, IgA, IgG1,IgG2, IgG3, IgG4, IgA1, IgA2, and combinations thereof.
 9. A method fordetecting the presence of at least two analytes in a patient's singlefluid sample, the method comprising the steps of: upon receipt of apatient's single fluid sample which may comprise at least two analytes,placing the patient's single fluid sample in a sample receptacle;drawing the single fluid sample into a first pipette that is disposedwithin the sample receptacle, wherein a first immobilized bindingpartner is present on an inner surface of the first pipette and thefirst immobilized binding partner is brought into contact with thewithdrawn single fluid sample such that a first analyte-binding partnercomplex is formed if a first analyte is present in the single fluidsample; re-dispensing a non-reacted portion of the withdrawn singlefluid sample from the first pipette and into the fluid samplereceptacle; drawing at least a portion of the re-dispensed fluid sampleinto a second pipette that is disposed within the sample receptacle,wherein a second immobilized binding partner is present on an innersurface of the second pipette, the second immobilized binding partnercomprising an analyte specificity different from that of the firstimmobilized binding partner, further wherein the second immobilizedbinding partner is brought into contact with the re-dispensed fluidsample such that a second analyte-binding partner complex is formed if asecond analyte is present in the re-dispensed fluid sample; anddetecting the presence of the first analyte-binding partner complex andthe presence of the second analyte-binding partner complex.
 10. Themethod of claim 9, wherein the patient's single fluid sample is selectedfrom the group consisting of whole blood, blood plasma, blood serum,saliva, sputum, cerebrospinal fluid (CSF), intestinal fluid,intraperotineal fluid, cystic fluid, sweat, interstitial fluid, tears,mucus, urine, bladder wash, and semen.
 11. The method of claim 9,wherein the patient's single fluid sample comprises a volume from about1 microliter to about 100 microliters.
 12. The method of claim 11,wherein the volume of the patient's single fluid sample is about 50microliters.
 13. The method of claim 9, wherein the sample receptacle isselected from the group consisting of a well, cuvette, tube, vial, andcapillary.
 14. The method of claim 9, wherein the first analyte andsecond analyte are selected from the group consisting of cardiacmarkers, infectious disease serological analytes, therapeutic drugs,drugs of abuse, hormones, cancer markers, infectious disease nucleicacid analytes, autoimmune disease nucleic acid markers, proteins,vitamins, cofactors, metabolites, and combinations thereof.
 15. Themethod of claim 9, wherein the first immobilized binding partner and thesecond immobilized binding partner are selected from the groupconsisting of intact monoclonal antibodies, polyclonal antibodies,multi-specific antibodies, bispecific antibodies, antibody fragments,receptors, ligands, aptamers, antigens, antibody substitute proteins andpeptides, molecular imprinted polymers, and combinations thereof. 16.The method of claim 15, wherein the first immobilized binding partnerand the second immobilized binding partner are selected from the groupconsisting of IgG, IgE, IgM, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1,IgA2, and combinations thereof.
 17. The method of claim 9, wherein thedetection of the first analyte and the second analyte is conducted viachemiluminescent detection, fluorescent detection, and combinationsthereof.
 18. A kit for detecting the presence of at least two analytesin a patient's single fluid sample, the kit comprising: at least twopipettes, wherein the at least two pipettes comprises a first pipettehaving a first immobilized capture antibody specific to a first analytepresent on an inner surface of the first pipette and a second pipettehaving a second immobilized capture antibody specific to a secondanalyte present on an inner surface of the second pipette, wherein theanalyte specificity of the first immobilized capture antibody isdifferent than the analyte specificity of the second immobilized captureantibody; a fluid sample receptacle, wherein the fluid sample receptacleis configured to hold a patient's single fluid sample; and providinginstructions to a user to allow the user to use the kit to identify thepresence of at least two analytes in the single fluid sample.